Release and modulation of release of serotonin in rabbit superior colliculus.

The release of previously incorporated [3H]serotonin and its presynaptic modulation were studied in slices of rabbit superior colliculus. Electrical stimulation at frequencies of 0.017-3 Hz greatly increased the outflow of tritiated compounds; this response was almost abolished by tetrodotoxin and in a low calcium medium. Unlabelled serotonin, when added in the presence of nitroquipazine, an inhibitor of high-affinity neuronal serotonin uptake, reduced the electrically evoked overflow of tritium, an effect antagonized by metitepin. Given alone, metitepin caused an increase. The evoked overflow was also decreased by clonidine, and the effect of clonidine was counteracted by phentolamine. Phentolamine itself increased the overflow response. However, this was probably not due to antagonism against an inhibitory effect of endogenous noradrenaline because, first, the selective alpha 2-adrenoceptor antagonist idazoxan did not share with phentolamine the overflow-enhancing effect, second, phentolamine continued to increase the overflow after noradrenergic axons had been destroyed by 6-hydroxydopamine, and third, the facilitatory effects of metitepin and phentolamine were not additive. Phentolamine, like metitepin, antagonized the presynaptic inhibitory effect of serotonin, indicating that it may increase the evoked overflow of tritium by blocking serotonin receptors rather than alpha-adrenoceptors. Ethylketocyclazocine decrease the electrically evoked overflow, and its effect was prevented by naloxone: peptides selective for opioid mu- or delta-receptors caused no change. Nicotine increased the basal outflow of tritium (in the absence of electrical stimulation); the increase was attenuated by hexamethonium and low calcium medium. No or minimal changes in tritium outflow were obtained with beta-adrenoceptor, dopamine receptor, muscarine receptor and GABA receptor ligands or with substance P and glutamate. In conjunction with our previous studies, these results indicate that serotonin is a neurotransmitter in the superior colliculus. Its release is modulated through presynaptic autoreceptors (probably 5-HT1), alpha 2-adrenoceptors, opioid kappa-receptors and nicotine receptors, of which only the autoreceptors receive an endogenous input, at least under the experimental conditions chosen. Each of the three groups of collicular monoamine axons that we have studied recently (cholinergic, noradrenergic, serotoninergic) possesses a specific pattern of presynaptic, release-modulating receptors. A physiological role seems likely only for the alpha 2-autoreceptors at the noradrenergic and the 5-HT1-autoreceptors at the serotoninergic axons.

Subclassification of the presynaptic alpha 2-autoreceptors in rabbit brain cortex.

1. alpha 2-Adrenoceptor binding sites have been subclassified into alpha 2A sites of which a main characteristic is very low affinity for prazosin, and alpha 2B sites with relatively high affinity for prazosin. The presynaptic alpha 2-autoreceptors in rabbit brain cortex were studied in order to classify them in terms of alpha 2A and alpha 2B. Release of [3H]-noradrenaline in cortical slices was elicited by trains of 4 pulses delivered at 100 Hz. 2. Clonidine caused concentration-dependent inhibition of the stimulation-evoked overflow of tritium, with an EC50 of 7.5 nM and a maximal inhibition by 96%. 3. The following alpha-adrenoceptor antagonists shifted the concentration-response curve of clonidine to the right (antagonist-receptor dissociation constants KD in brackets): yohimbine (14 nM), 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazo le (BRL 44408; 15 nM) and 1,2-dimethyl-2,3,9,13betetrahydro-1H-dibenzo[c,f]imidazo[1,5-a]aze pine (BRL 41992; 630 nM). Prazosin 1 microM and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]-ethyl]-4,4-dimethyl-1,3 (2H,4H)-isoquinolinedione (AR-C 239) 1 microM failed to antagonize the effect of clonidine. Higher concentrations of prazosin and AR-C 239 greatly accelerated the basal efflux of tritium. 4. The method used permits the functional determination of antagonist affinities undistorted by endogenous alpha 2-autoinhibition. A comparison with affinities derived from radioligand binding experiments indicates that the presynaptic alpha 2-autoreceptors in rabbit brain cortex are markedly different from the alpha 2B-subtype and probably belong to the prazosin-insensitive alpha 2A-subtype.

Pharmacological characterization of presynaptic alpha 2-autoreceptors in rat submaxillary gland and heart atrium.

1. The pharmacological properties of presynaptic alpha 2-autoreceptors were studied in rat isolated submaxillary glands and atria. Tissue pieces were preincubated with [3H]-noradrenaline, then superfused with medium containing desipramine, and stimulated electrically. In one series of experiments, pEC30 values of 12 alpha-adrenoceptor antagonists were determined, i.e., negative logarithms of concentrations that increased the electrically evoked overflow of tritium by 30%. In another series, pKD values of 9 alpha-adrenoceptor antagonists against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), and of 3 antagonists against the release-inhibiting effect of methoxamine, were determined. 2. In submaxillary glands, the pEC30 values of the antagonists correlated well with their pKD values against UK 14304 (r = 0.93). The same was true for atria (r = 0.92). 3. In submaxillary glands, the pKD values of 3 antagonists against UK14304 were very similar to their pKD values against methoxamine, with a maximal difference of 0.4. The same was true for atria where the maximal difference was 0.3. 4. The pEC30 values obtained in submaxillary glands correlated significantly with those obtained in atria (r = 0.81). The same was true for the pKD values (r = 0.79). However, the pEC30 and pKD values also indicated consistent differences between the two tissues. 5. It is concluded that the sites of action of the imidazoline UK 14304 (alpha 2-selective), the phenylethylamine noradrenaline, and the phenylethylamine methoxamine (alpha 1-selective) are exclusively alpha 2-adrenoceptors. There is no indication for presynaptic alpha 1-adrenoceptors or for an effect of UK 14304 mediated by presynaptic imidazoline receptors.The 02-autoreceptor population in the submaxillary gland differs from that in the atrium.6. Comparison with studies from the literature indicates that the submaxillary autoreceptors are closely similar to the a2D radioligand binding site found in the bovine pineal gland and probably the rat submaxillary gland. The atrial autoreceptors also conform best to this site, but the agreement is more limited; the atrial autoreceptors may represent a type related to, but distinct from, the a2D site, or a mixture of different types.

Alpha 2C-adrenoceptor-modulated release of noradrenaline in human right atrium.

1. The aim of the present study was to characterize the presynaptic alpha 2-autoreceptors in human right atrium in terms of the alpha 2A-D system. Segments of atrial appendages were preincubated with [3H]-noradrenaline and then superfused in the presence of cocaine and stimulated electrically. pEC30% values of eight alpha-adrenoceptor antagonists with discriminatory power were determined. pEC30% is the negative logarithm of the antagonist concentration that increased the stimulation-induced overflow of tritium by 30%. For four antagonists, the dissociation constant KD was determined, in addition to pEC30%, against the overflow-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under autoinhibition-free conditions. 2. pEC30% and KD values yielded identical rank orders of antagonist affinity (rauwolscine > WB 4101 > phentolamine > prazosin) suggesting that both released noradrenaline and the exogenous agonist UK 14,304 activated the same receptor to inhibit release. 3. The eight antagonist pEC30% values obtained in right atrium correlated significantly with their pEC30% values, reported in the literature, at the presynaptic alpha 2C-autoreceptors in human kidney (r = 0.817; slope of the regression line 1.03). No significant correlation was obtained between pEC30% values at atrial autoreceptors and pKD values at previously characterized alpha 2A-autoreceptors in rabbit and alpha 2D-autoreceptors in rat, mouse and guinea-pig tissues. 4. Comparison of antagonist pEC30% values with their pKD values at native alpha 2 binding sites in cells or tissues that express a single subtype only, and with pKD values at alpha 2 binding sites in membranes of COS cells transfected with human alpha 2 subtype genes confirms the alpha 2C character of the atrial autoreceptors: significant correlations were obtained exclusively with the alpha 2C binding sites. 5. Ratios of KD values were computed for alpha 2-autoreceptors in human right atrium and for binding sites in COS cells transfected with human alpha 2 subtype genes. The autoreceptor ratios corresponded well with the respective ratios for the alpha 2C binding sites (maximal three fold deviation) but were, in part, markedly different from ratios calculated for alpha 2A and alpha 2B binding sites (up to 166 fold deviation). This outcome supports the alpha 2C designation of the autoreceptors. 6. In conclusion, the presynaptic alpha 2-autoreceptors in human right atrium are alpha 2C. In this they agree with the previously characterized alpha 2-autoreceptors in human kidney. The alpha 2C classification possibly separates, in general, human alpha 2-autoreceptors from those in lagomorph (rabbit) and rodent (rat, mouse, guinea pig) species that have been proposed to be predominantly alpha 2A or alpha 2D.

The transmitter release pattern of serotonin axons in rabbit brain cortex slices during short pulse trains.

The release of serotonin was studied in superfused rabbit brain cortex slices that had been preincubated with [(3)H]serotonin. The slices were stimulated by single electrical pulses or by trains of 4 pulses delivered at 0.011, 1 or 100 Hz. The overflow of tritium elicited by these stimuli was calcium-dependent and tetrodoxin- and Mg(2+)-sensitive. When the superfusion medium contained nitroquipazine or fluvoxamine, the overflow elicited by 4 pulses at 0.011 or 1 Hz was about 2-fold, whereas the overflow elicited by 4 pulses/100 Hz was about 3.7-fold the single pulse-evoked overflow. Metitepin increased little the overflow of tritium elicited by single pulses or 4 pulses/100 Hz, but increased by up to about 150% the overflow evoked by 4 pulses at 1 or 0.011 Hz. Unlabelled serotonin inhibited the evoked overflow in a similar manner, irrespective of the stimulation conditions. When the superfusion medium did not contain serotonin uptake inhibitors, the overflow elicited by a single pulse was too low to be reliably measured. Metitepin increased only slightly the overflow at 4 pulses/0.011 Hz or 4 pulses/100 Hz but increased by up to about 160% the overflow at 4 pulses/1 Hz. The results indicate that the release of serotonin elicited by single pulses as well as by 4 pulses/100 Hz is subject to only a small tonic, stimulation-independent presynaptic autoinhibition, and under these conditions the three pulses following the first one at intervals of 10 ms release about the same amount of transmitter as does pulse No. 1. In contrast, stimulation-dependent presynaptic autoinhibition develops in trains of 4 pulses delivered at 0.011 Hz (only when serotonin re-uptake is blocked) or 1 Hz so that there is a marked fall in the release elicited by each pulse in the course of the train. The total release elicited by such short, autoinhibited trains is dominated by the large response to pulse No. 1. It seems possible that more presynaptic autoreceptors are available for exogenous agonists than for released serotonin.

Further study of prerequisites for the enhancement by alpha-adrenoceptor antagonists of the release of noradrenaline.

Segments of the rabbit ear artery were preincubated with (-)-3H-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of 3H-noradrenaline and total tritium was determined. In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 microM present). Thirteen pulses (0.25 Hz) elicited an overflow of 3H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 microM did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 microM decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 microM decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 microM was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses. In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of 3H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 microM did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial agonist effect of 2-[2-(1,4-benzodioxanyl)]-2-imidazoline (RX 781 094) at presynaptic alpha 2-adrenoceptors in rabbit ear artery.

Ear arteries from rabbits were preincubated with 3H-noradrenaline and then perfused. 2-[2-(1,4-Benzodioxanyl)]-2-imidazoline (RX 781094) 0.1 or 1 microM reduced the overflow of 3H-noradrenaline and total tritium elicited by 13 electrical pulses at 0.25 Hz or 26 Pulses at 0.5 Hz. RX 781 094 1 microM increased the overflow elicited by 52 pulses at 1 Hz. The inhibitory effects were blocked by yohimbine 10 microM but not by prazosin 1 microM. The alleged antagonist RX 781 094 possesses intrinsic activity at the presynaptic alpha 2-autoreceptors of the ear artery.

Presynaptic alpha 2-adrenoceptor, opioid kappa-receptor and adenosine A1-receptor interactions on noradrenaline release in rabbit brain cortex.

The interaction of presynaptic, release-inhibiting alpha 2-adrenoceptors, opioid kappa-receptors and adenosine A1-receptors was studied in slices of the occipito-parietal cortex of the rabbit. The slices were preincubated with 3H-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S1, S2). The stimulation-evoked overflow of tritium was taken to reflect action potential-evoked release of noradrenaline. One of two release-modulating compounds to be examined for interaction was kept in the medium throughout superfusion, the other one was added before S2. In many experiments, the stimulation parameters were adjusted (frequency 0.5-7 Hz; voltage drop 2-5 V/cm) in order to obtain similar reference release (S1) values despite the presence of the first release-modulating compound. The selective kappa-receptor agonist ethylketocyclazocine (EK) attenuated markedly the release-inhibiting effects of the alpha 2-adrenoceptor-selective agonists clonidine and alpha-methylnoradrenaline as well as the release-facilitating effect of the alpha 2-adrenoceptor-selective antagonist yohimbine. The attenuation occurred both when the parameters of electrical stimulation were kept constant and when they were adjusted to obtain similar S1 release values. The selective A1-receptor agonist R-N6-phenylisopropyladenosine (PIA) also attenuated the effects of clonidine and yohimbine. Conversely, clonidine attenuated and yohimbine enhanced the release-inhibiting effect of PIA. Yohimbine also enhanced the release-facilitating effect of the adenosine receptor antagonist 8-phenyltheophylline. Again, these changes occurred both at constant stimulation parameters and when stimulation parameters were adjusted.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmitter release patterns of noradrenergic, dopaminergic and cholinergic axons in rabbit brain slices during short pulse trains, and the operation of presynaptic autoreceptors.

Slices of rabbit brain were field-stimulated either by single electrical pulses or by trains of 4 or 8 pulses at 1 or 100 Hz in order to study transmitter release patterns and the autoinhibition of transmitter release. The slices were preincubated with 3H-noradrenaline (cortex), 3H-dopamine (caudate nucleus) or 3H-choline (caudate nucleus). Slices preincubated with 3H-noradrenaline were superfused with medium containing desipramine 1 mumol/l. The overflow of tritium elicited by single pulses amounted to 0.19% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was similar, whereas that elicited by 4 pulses/100 Hz was 5.1-fold higher. Yohimbine 10-1000 nmol/l increased up to 2.5-fold the overflow evoked by 4 pulses/1 Hz but did not change the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with 3H-dopamine were superfused with medium containing nomifensine 1 mumol/l. The overflow of tritium elicited by single pulses was 0.39% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was 1.3-fold and the overflow elicited by 4 pulses/100 Hz 1.4-fold higher. Domperidone 1-100 nmol/l and sulpiride 10-1000 nmol/l increased up to 2.4-fold the overflow evoked by 4 pulses/1 Hz but increased only slightly the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with 3H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 mumol/l. In physostigmine-free medium, atropine did not increase the evoked overflow of tritium at any stimulation condition. In physostigmine-containing medium, the overflow elicited by single pulses was 0.18% of the tritium content of the tissue. The overflow elicited by 8 pulses/1 Hz was 2.0-fold and the overflow elicited by 8 pulses/100 Hz 2.2-fold higher. Atropine 2-200 nmol/l increased up to 2.4-fold the overflow evoked by 8 pulses/1 Hz but increased only slightly the overflow evoked by single pulses or 8 pulses/100 Hz. In physostigmine-free medium, sulpiride 10-1000 nmol/l did not change the single-pulse-evoked overflow of tritium in the absence but increased it in the presence of nomifensine 1 mumol/l. Single pulses elicit a large release of 3H-noradrenaline, 3H-dopamine and 3H-acetylcholine under the conditions of these experiments. Release elicited by single pulses is not subject to autoinhibition except for a small inhibition by spontaneously released transmitter in the case of dopaminergic and cholinergic axons.(ABSTRACT TRUNCATED AT 400 WORDS)

Mathematical analysis of the control of neurotransmitter release by presynaptic receptors as a supplement to experimental data.

The quantitative analysis of receptor-mediated effects is based on experimental concentration-response curves in which an independent variable, the concentration of a receptor ligand, is linked with a dependent variable, the biological response. The steps that intervene between the ligand-receptor interaction and the subsequent biologic effect, i.e. modulation of transmitter release in our examples, are largely unknown. Nevertheless, the shape of a concentration-response curve may give some insights into the nature of the relation between receptor occupancy and ensuing response. The shape of the concentration-response curve can be evaluated by nonlinear regression analysis of the data points of the independent and dependent variable. If possible, the model applied should be mechanistically derived from a physical or chemical law, underlying the biological condition. For instance, the inherence of the Law of Mass Action allows to call the model mechanistic. The presence of spare receptors for an agonist must induce an alteration of the shape of the concentration-response curve as compared to a symmetric bimolecular concentration-binding curve. Evaluation methods which neglect the alteration of the geometrical form of concentration-response curves due to non-proportionality between receptor occupation and relative response do not seem appropriate to quantify spare receptors. The "general response function" may allow a mechanistic interpretation of the occupancy-response relationship. This function estimates roughly the number of "non-spare" receptors and of spare receptors on a functional unit that contribute to the response.

Species differences in presynaptic serotonin autoreceptors: mainly 5-HT1B but possibly in addition 5-HT1D in the rat, 5-HT1D in the rabbit and guinea-pig brain cortex.

The pharmacological properties of presynaptic serotonin autoreceptors were compared in slices of rat, rabbit, and guinea-pig brain cortex. The slices were preincubated with 3H-serotonin and then superfused with medium containing fluvoxamine 3 mumol/l and stimulated four times by trains of four pulses delivered at 100 Hz. Cumulative concentration-response curves were determined and used for the calculation of agonist EC50 values and maximal effects and antagonist KB values. Unlabelled serotonin itself and the serotonin receptor agonists 5-carboxamidotryptamine (5-CT), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) and (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced the stimulation-evoked overflow of tritium with a rank order of potency 5-CT = RU 24969 greater than serotonin greater than 8-OH-DPAT in the rat and 5-CT greater than serotonin greater than RU 24969 greater than 8-OH-DPAT in the rabbit and guinea-pig. Ipsapirone caused no change. Metitepine and metergoline antagonized the effect of 5-CT; the KB values were lower in the rabbit and guinea-pig than in the rat. Yohimbine at up to 1 mumol/l did not reduce the evoked overflow of tritium and did not antagonize the inhibitory effect of 5-CT in the rat but reduced the evoked overflow in the rabbit and counteracted the effect of 5-CT in the guinea-pig. (-)-Propranolol, conversely, reduced the evoked overflow of tritium in the rat but neither reduced the evoked overflow nor antagonized the effect of 5-CT in the rabbit and guinea-pig. Isamoltane did not significantly change the effect of 5-CT in any species. In the rat, it also failed to antagonize the inhibitory effect of 8-OH-DPAT but did antagonize the effect of RU 24969. The inhibition caused by 8-OH-DPAT persisted in the presence of idazoxan but was attenuated by metitepine in all species. The experimental conditions used permit the determination of the constants of agonist and antagonist action undistorted by autoinhibition. The results confirm the view that the serotonin axons of rat brain possess 5-HT1B autoreceptors. They show by direct comparison under identical conditions that the autoreceptors in rabbit and guinea-pig are very similar to each other but differ markedly from those in the rat. The results give additional credence to previous suggestions that, in the rabbit and guinea-pig, the autoreceptors are 5-HT1D. The serotonin axons of rat brain cortex may possess 5-HT1D in addition to 5-HT1B autoreceptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Protection of presynaptic alpha-adrenoceptors against irreversible blockade by phenoxybenzamine: preservation of the modulatory effects of exogenous noradrenaline and yohimbine.

1. Receptor protection experiments were carried out in order to study the site of action of alpha-adrenoceptor agonists and antagonists on the release of noradrenaline. Cerebrocortical slices from rabbits were preincubated with 3H-noradrenaline. They were then superfused with medium containing cocaine 30 mumol/l and stimulated electrically (3 Hz) three times, after 60, 250 and 295 min of superfusion (S1, S2, S3). Phenoxybenzamine 10 mumol/l, when used, was added between S1 and S2 for 30 min; putative protecting drugs (clonidine 100 mumol/l or yohimbine 10 mumol/l) were present 5 min before and during the exposure to phenoxybenzamine and then washed out together with the latter. Either the voltage drop between the electrodes at S2 and S3 or the Ca2+ -concentration of the superfusion medium at S2 and S3 was diminished, if necessary, in order to bring the overflow evoked by S2 close to the overflow at S1. Blockade by phenoxybenzamine, or protection against the blockade, was examined by addition of the test compounds noradrenaline 0.1 mumol/l or yohimbine 1 mumol/l before S3. 2. In slices not exposed previously to alpha-adrenoceptor ligands, noradrenaline 0.1 mumol/l greatly reduced, whereas yohimbine 1 mumol/l greatly increased the evoked overflow of tritium. Both effects were abolished in slices treated with phenoxybenzamine 10 mumol/l alone between S1 and S2. 3. In contrast to phenoxybenzamine alone, exposure to phenoxybenzamine 10 mumol/l in the presence of either clonidine 100 mumol/l or yohimbine 10 mumol/l failed to abolish the effects of the test compounds noradrenaline 0.1 mumol/l and yohimbine 1 mumol/l, although the effects were reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Only activated but not non-activated presynaptic alpha 2-autoreceptors interfere with neighbouring presynaptic receptor mechanisms.

Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic alpha 2-autoreceptors of effects mediated by presynaptic opioid kappa- and adenosine A1-receptors requires activation of the alpha 2-receptors. The slices were preincubated with 3H-noradrenaline and then superfused with medium containing desipramine 1 mumol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz. The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca2+-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 mumol/l or yohimbine 0.3 mumol/l. Ethylketocyclazocine (EK; 0.1-10 nmol/l) and R-(-)-N6-phenylisopropyladenosine (PIA; 1-1,000 nmol/l) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. - The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 mumol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca2+ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine. The results demonstrate that there is no alpha 2-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic alpha 2-adrenoceptor does not interfere with presynaptic opioid kappa- and adenosine A1-receptor mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic alpha 2-autoreceptors in brain cortex: alpha 2D in the rat and alpha 2A in the rabbit.

Presynaptic alpha 2-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [3H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz. The alpha 2-adrenoceptor agonist bromoxidine (UK 14304) reduced the electrically evoked overflow of tritium with EC50 values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazo le (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)aze pine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant KD. The KD values correlated only weakly between the rat and the rabbit. Dissociation constants KA of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the alpha 2-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The KA value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit. The results confirm the species difference between rat and rabbit brain presynaptic alpha 2-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the alpha 2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the alpha 2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of alpha 2-RG20, the putative rat alpha 2D-adrenoceptor gene (r = 0.97; P < 0.01), but not with their binding affinities for the gene product of alpha 2-C10, the putative human alpha 2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the alpha 2-C10 (r = 0.98; P < 0.001) but not with the alpha 2-RG20 gene product. Since presynaptic alpha 2-autoreceptors are also alpha 2D in rat submaxillary gland and perhaps vas deferens and alpha 2A in rabbit pulmonary artery, the possibility arises that the majority of alpha 2-autoreceptors generally are alpha 2D in the rat and alpha 2A in the rabbit. Moreover, receptors of the alpha 2A/D group generally may be the main mammalian alpha 2-autoreceptors.

Presynaptic alpha 2A-adrenoceptors inhibit the release of endogenous dopamine in rabbit caudate nucleus slices.

alpha 2-Adrenoceptors modulating the release of dopamine were identified and characterized in slices of the head of the rabbit caudate nucleus. Release of endogenous dopamine was measured by fast cyclic voltammetry as the increase in the extracellular concentration of dopamine elicited by electrical stimulation. The electrochemical signal was identified as dopamine by means of the oxidation potential, the voltammogram and the fact that the signal was not changed by desipramine, which inhibits the high affinity uptake of noradrenaline, but was greatly increased by nomifensine, which in addition inhibits the high affinity uptake of dopamine. Stimulation by 6 pulses/100 Hz increased the extracellular concentration of dopamine by about 85 nM. The selective alpha 2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced this release with an EC50 of 173 nM and by maximally 75%. The alpha 2-adrenoceptor agonists clonidine and oxymetazoline only tended to cause a decrease. Six drugs, including oxymetazoline, were tested as antagonists against UK 14,304. Their order of antagonist potency (pKD values in brackets) was rauwolscine (8.0) > oxymetazoline (7.5) > 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101; 7.3) > phentolamine (7.1) > corynanthine (5.1) approximately prazosin (<6). Given alone, the antagonists did not change the release of dopamine elicited by 6 pulses/100 Hz, and the same was true for the dopamine receptor antagonist sulpiride. When caudate slices were stimulated by 10 pulses/1 Hz, sulpiride increased the release of dopamine. Desipramine and rauwolscine, in contrast, again caused no change. It is concluded that dopaminergic axons in the rabbit caudate nucleus possess release-inhibiting alpha 2-adrenoceptors.2+ off

Mutual interaction between presynaptic alpha 2-adrenoceptors and opioid kappa-receptors at the noradrenergic axons of rabbit brain cortex.

The interaction between presynaptic, release-inhibiting alpha 2-adrenoceptors and opioid receptors was studied in slices of the parieto-occipital cortex of rabbits. The slices were preincubated with 3H-noradrenaline and then superfused with 3H-noradrenaline-free medium and stimulated electrically (3 or 7 Hz, 2 or 5 V/cm voltage drop between the electrodes). Clonidine and ethylketocyclazocine (EK) depressed, whereas yohimbine increased the electrically evoked overflow of tritium. When clonidine was administered first and retained in the medium for the rest of the experiment, the overflow-inhibiting effect of EK was reduced. When yohimbine was administered first and kept for the rest of the experiment, the effect of EK was enhanced. When, finally, EK was administered first and clonidine as the second drug, the overflow-inhibiting effect of clonidine was attenuated. The changes in the effect of EK (by clonidine or yohimbine) and clonidine (by EK) were not due to the changes in release per se produced by the drugs that were given first. Naloxone shifted the concentration-response curve of EK to the right; the dissociation constant of the naloxone-receptor complex, calculated from the shift, was 13 nmol/l. It is concluded that there is an interaction between presynaptic alpha 2-adrenoceptors and opioid kappa-receptors, either at the level of the receptors themselves or of the post-receptor reaction chains. Activation of one kind of receptor blunts the inhibition of release produced by activation of the other kind of receptor.

Serotonin uptake blockers influence serotonin autoreceptors by increasing the biophase concentration of serotonin and not through a "molecular link".

The mechanism of the attenuation, by serotonin uptake blockers, of the release-inhibiting effect of exogenous serotonin autoreceptor agonists was studied in rabbit brain cortex and rat hypothalamus slices. The slices were preincubated with 3H-serotonin and then superfused and stimulated electrically. In rabbit brain slices stimulated by trains of 4 pulses at 100 Hz, 5-carboxamidotryptamine and 5-methoxytryptamine reduced the evoked overflow of tritium, and their concentration-response curves were not changed by any of three serotonin uptake inhibitors, namely citalopram, fluvoxamine and 6-nitroquipazine. In contrast, when the slices were stimulated by trains of 10 pulses at 0.033 Hz, fluvoxamine shifted the concentration-response curve of 5-methoxytryptamine to the right. Experiments with the autoreceptor antagonist metitepine indicated that little, if any, endogenous autoinhibitory tone developed in the course of trains of 4 pulses/100 Hz, irrespective of the absence or presence of uptake inhibitors, as well as during trains of 10 pulses/0.033 Hz in the absence of uptake inhibitors, whereas marked autoinhibition developed when 10 pulses/0.033 Hz were applied in the presence of fluvoxamine. In rat hypothalamic slices stimulated by trains of 4 pulses at 100 Hz, citalopram also failed to change the concentration-response curve of 5-methoxytryptamine. These results indicate that serotonin uptake blockers attenuate the effect of exogenous autoreceptor agonists by an increase in the biophase concentration of released serotonin and, hence, in endogenous autoinhibitory tone, and not by some direct "molecular link" unrelated to the biophase concentration of released serotonin.

Subclassification of presynaptic alpha 2-adrenoceptors: alpha 2D-autoreceptors in mouse brain.

The study was devised to classify, by means of antagonist affinities, the presynaptic alpha 2-autoreceptors in mouse cerebral cortex in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Slices of the cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. The stimulation periods used (4 pulses, 100 Hz) did not lead to alpha 2-autoinhibition as shown by the lack of an increase by rauwolscine of the evoked overflow of tritium. The alpha 2-selective agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and alpha-methylnoradrenaline reduced the evoked overflow. All 10 antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. Rauwolscine also shifted the concentration-inhibition curve of alpha-methylnoradrenaline to the right. pKd values of the antagonists were calculated from the shifts. The pKd values of rauwolscine against UK 14,304 and alpha-methylnoradrenaline were very similar (8.0 and 7.9, respectively). Comparison with antagonist affinities for prototypic native alpha 2 binding sites, alpha 2 binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that the alpha 2-autoreceptors in mouse brain cortex are alpha 2D. This is the first subtype determination of alpha 2-autoreceptors in the mouse. It supports the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D branch of the alpha 2-adrenoceptor tree.

Subclassification of presynaptic alpha 2-adrenoceptors: alpha 2D-autoreceptors in guinea-pig atria and brain.

The study was devised to classify, by means of antagonist and agonist affinities, the presynaptic alpha 2-autoreceptors in guinea-pig heart atria and brain cortex in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D. A set of antagonists and agonists was chosen that was able to discriminate between the four subtypes. Small pieces of the atria and slices of the brain cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. In one series of experiments (atria only), tissue pieces were stimulated by relatively long pulse trains (1 min) leading to marked alpha 2-autoinhibition. All 10 antagonists increased the evoked overflow of tritium. pEC30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments (atria and brain slices), tissue pieces were stimulated by brief pulse trains (0.4 s or 40 ms) that led to little (atria) or no (brain slices) alpha 2-autoinhibition, and antagonist effects against the alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2- ylamino)-quinoxaline (UK 14,304) were examined. All 10 (atria) or 8 (brain) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. In a third series of experiments (brain slices only), also with brief pulse trains (40 ms), pKa values (negative logarithms of dissociation constants of agonist-alpha 2-adrenoceptor complexes) were determined by comparison of concentration-inhibition curves of UK 14,304, guanoxabenz and oxymetazoline in normal tissue and in tissue in which a fraction of the receptors had been blocked by phenoxybenzamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular effects of agmatine, a "clonidine-displacing substance", in conscious rabbits.

Agmatine has been identified as a "clonidine-displacing substance" in extracts from bovine brain. We studied its effect on cardiovascular regulation and the role played in this effect by alpha 2-adrenoceptors. In conscious rabbits, agmatine 10 micrograms kg-1 injected intracisternally (i.c.) caused no change, whereas agmatine 30, 100 and 300 micrograms kg-1 i.c. increased renal sympathetic nerve firing, the plasma concentration of noradrenaline and adrenaline and arterial blood pressure. Heart rate tended to be decreased. Yohimbine 1.5 micrograms kg-1 i.c. caused no change, whereas yohimbine 5, 15 and 50 micrograms kg-1 increased renal sympathetic nerve activity, the plasma concentration of noradrenaline and adrenaline, blood pressure and heart rate. In rabbit brain cortex slices preincubated with [3H]-noradrenaline, agmatine 1 to 100 microM did not modify the electrically evoked overflow of tritium (either 4 pulses at 100 Hz or 36 pulses at 3 Hz). The evoked overflow was reduced by 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304) 0.03 to 30 nM (4 pulses at 100 Hz), and this inhibition was not affected by agmatine 10 and 100 microM. Agmatine did not change the basal efflux of tritium. The results show that agmatine, like yohimbine, causes central sympathoexcitation when given i.c., but agmatine differs from yohimbine in that it does not increase heart rate. Agmatine acts neither as an agonist nor as an antagonist at the alpha 2-autoreceptors in rabbit brain cortex. alpha 2-Adrenoceptors, therefore, are probably not involved in its cardiovascular effects.(ABSTRACT TRUNCATED AT 250 WORDS)